6-deoxy-6-demethyl-6-halomethylene tetracyclines and their 11a-chloro and fluoro derivatives



United States Patent 0 19 Claims. (Cl. 260-559) This application is adivisional application of application Serial No. 221,870 as filedSeptember 6, 1962, now US. Patent No. 3,183,267, issued May 11, 1965,said latter application being a continuation-in-part of now abandonedapplication Serial No. 72,875, filed December 1, 1960, which in turn isa continuation-in-part of abandoned application Serial No. 31,236, filedMay 23, 1960.

This invention relates to new and useful antibacterial agents of thetetracycline type, salts thereof and processes for their production, asWell as intermediates useful therefor.

More particularly, this invention relates to new and usefultetracyclines of the following formulae:

t CONH:

I I A (H X, /OHH:C ONE:

Y I I iodo and nitro;

X is selected from the group consisting of hydrogen, nitro and amino;

3,250,809 Patented May 10, 1966 X is selected from the group consistingof hydrogen and chloro; and

X is selected from the group consisting of chloro, bromo,

iodo, nitro and amino.

The present new compounds are hereinafter referred to as6-deoxy-6-demethyl-6-methylenetetracyclines. These new compounds areuseful by virtue of their high antibacterial activity and also asintermediates for the preparation of compounds of high antibacterialactivity, as hereinafter described.

The present new compounds are prepared by 1151- dehalogenation ofIla-halo compounds of the formulae:

r X is selected from the group consisting of chloro, bromo I 3 Compoundsof Formulae 1V, V, VI and VII are prepared according to the followingsequence of reactions:

V q 4 a diethylene glycol (diglyme) and the dimethyl ether of ethyleneglycol (monoglyme). Temperature does not ap- N(OH:J2

on o Y 0H 0 xv (VIII) x, on, N(CH3)2 X: on, Mona):

OH A OH I 0151K OHlI OzN CONE: CONHI and Y2; 1 all Ya Y (XII) (IX) lCHNO: N(CHa)a CHX 'N(CH3)2 I /K 7 on a l CONH: CONH: .sHll' t am... a

(XIII) (X) l n): mom aux N(CH3)2 on: on! x1 Y coma, 0m g AJQNH,

' (IDH 6Y6 A SH 6y 0 (l (XIV) (XI) In the above sequence, X and X arechloro, iodo or bromo; X is hydrogen or chloro; and Y is chloro orfluoro, provided that when X; is chloro, Y is chloro.

The above sequence of reaction may be summarized as follows:

XV- VIII, lla-halogenation;

VIII IX, treatment with a strong acid; IX X, and X XIV, halogenation; X-XI, and 1X XII and XIII, nitration.

The lla-halogenation reaction is accomplished by merely contacting theselected tetracycline starting compound, preferably in the amphotericform, with a halogenating agent'in a reaction-inert solvent. For theproduction of the present new lla-halotetracyclines in which the halogenis Cl, a variety of halogenating agents may be used, including chlorine,iodochloride, N-chloro-lower alkanoic acid amides, e.g.N-chloroacetarnide, hydrocarbon dicarboxylig acid imides, e.g.N-chlorosuccinimide, phthalirnide and the like, N-lower-alkanoylanilides; 3-chlor0, 3-bromo, and 3,5-dichloro-5,5-dimethylhydantoin,perchloride hydrohalides, e.g. pyridinium perchloride hydrochloride, andlower alkyl hypochlorites e.g. 3 butylhypochlorite. It is obvious that,in general, any chlorinating agent common 1y employed in the art isoperable, but the above are preferred.

By reaction-inert solvent as employed herein is meant a solvent which,under the conditions of the reaction, does not react in an undesiredmanner With either starting compounds or final products. A minimum oflaboratory experimenta'tion will permit the selection of suitablesolvents for the present process. Exemplary of such solvents aredioxane, tetrahydrofuran, acetone, the dimethyl ether of pear to becritical in this process, temperatures from 25 to 50 C. being foundsuitable. Temperatures above 50 C. should preferably be avoided, due tothe possible formation of 5a,6-anhydro compounds and other degradationproducts, with consequent reduction in yield. The selection of the bestreaction conditions, e.g. temperature, solvent, halogenating agent,etc., is a matter of routine experimentation.

The lla-chlorotetracycline hemi-ketals are preferably prepared using awater-miscible solvent system and the products obtained by waterdilution of the reaction mixture. Once formed, they are found to berelatively stable in acidic aqueous solutions. For example,Ila-chlorotetracyeline-6,IZ-hemi-ketal hydrochloride on standing inWater at pH of about 1 for 18 hours at room temperature shows nodetectable decomposition.

The preparation of lla-fluorotetracycline hemi-ketals is accomplished bycontacting tetracycline compounds with perchloryl fluoride in thepresence of a'base, preferably an alkali metal hydroxide or allroxide.The reaction is usually carried out by dissolving the starting compoundin the selected solvent containing at least a molar equivalent of thebase and adding perchloryl fluoride, a gas at room temperature, in theusual fashion. As the reaction proceeds, the pH of the solution dropsfrom alkaline to near neutral values, the product usually commencing toseparate-at a lected in the usual fashion and dried. A

pH of approximately 8. The crystalline product is col-v pound withperchloric acid or liquid hydrogen fluoride.

It is preferred to employ aqueous perchloric acid at a concentration of60-70%. Optimum reaction conditions are readily determined by routineexperimentation. In carrying out this process, the starting compound isadded to the selected acid and, in general, allowed to react forrelatively short periods of time. For example, the starting compound isadded to liquid hydrogen fluoride at to 50 C. and allowed to stand fortime periods of as little as to 7 minutes and up to several hours, afterwhich the hydrogen fluoride is allowed to evaporate. The residue is thentreated by standard procedures to obtain crystalline product as thehydrofluoride salt, e.g. stirred in a nonsolvent and recrystallized froma solvent such as a lower alkanol. When the starting compound for thisprocess is an lla-fluoro compound, excellent results are obtained usingperchloric acid. When perchloric acid is used with the lla-fluorocompounds, the reaction is preferably carried out at temperatures ofabout 50 C. and higher, preferably from about 60 C. to 70 C. More highlyconcentrated perchloric acid may be used in the process but its use isnot preferred because of the explosion hazard with which those skilledin the art are familiar. The reaction time does not appear to becritical since the reaction appears to be almost instantaneous. Forexample, a reaction time of from 5 to minutes is usually found to giveexcellent results when the reaction temperature is in the range of 60-70C. At lower temperatures, slightly longer reaction periods may berequired to obtain substantial yield of the product.

After the reaction is complete, the product is obtained by standardprocedures. For example, it is most convenient to merely dilute thereaction mixture with nonsolvent, e.g. water, ether and the like, whichresults in precipitation of the product as the salt corresponding to theacid used. The products may be converted to the free base or any desiredsalt in the usual manner.

Nitration is accomplished by any of the standard methods used for thispurpose. For example, the starting compound is reacted with nitric acidper se or formed in situ, e.g. potassium nitrate in anhydroushydrofluoric, fluoroacetic or sulfuric acid. The nitration reaction mayalso be carried out in a solvent, such as a lower alkanoic acid, e.g.acetic acid, although a variety of other solvents, including water, mayalso be used. A minimum of laboratory experimentation Will permit theselection of suitable solvents. While reaction may vary appreciably, theuse of high temperatures should be avoided. Satisfactory results areobtained over the range of from. about 0 C. to about 80 C., with roomtemperature preferred as most convenient for most solvents. Reactiontime is not overly critical, particularly in view of the stability ofthe starting compounds to the acid reaction conditions. To ensure bestyields it is preferred to use reaction periods of from about 6 to 24hours. Nitration in strong acid solvents such as trifluoroacetic acid orHF generally occurs on the 6-methylene substituent, although when diluteaqueous nitric acid or nitric acid in aqueous acetic acid is used, theprincipal product is the 9-nitro compound. The coproduced isomers areseparable from the principal products by standard techniques, forexample, fractional crystallization, column chromatography and/ orcountercurrent distribution using known solvent systems.

The direct halogenation is accomplished by merely contacting thestarting 1la-halo compound with a halogenating agent in a reaction-inertsolvent, preferably a strong acid such as trifluoroacetic acid oranhydrous hydrogen fluoride. A number of halogenating agents may be usedfor this purpose, including those enumerated hereinbefore as well ascorresponding bromo and iodo compounds, such as bromine,N-iodosuccinimide and the like. To obtain monohalo substitutionproducts, it is usually preferred to use an equimolar amount ofhalogenating agent i.e. brominating, chlorinating or iodinating agent,although excess amounts up to about mole percent may also be employed.To obtain dihalo compounds, a two molar ratio of halogenating agent isused. Alternatively, the mono-halo compounds may be further halogenatedwith an equimolar amount of reagent to obtain dihalo compounds. The timeof reaction will vary with the starting compound and the selectedhalogenating agent. Generally, the reaction is allowed to proceed untila negative starch-iodide test is obtained. Reaction temperatures rangingfrom 25 up to about C. may be used, althoughit is usually preferred toemploy temperatures ranging between 20 and 40 C. for reaction intrifluoroacetic acid, and 020 C. in liquid HF. When the reaction iscomplete, the product is obtained by any of the standard methods ofisolation, which include, for example, precipitation by concentration ofthe reaction mixture or by dilution 'with a non-solvent, e.g. ether,hexane. The product as obtained may be further purified or utilized incrude form for the lla-dehalogenation reaction hereinafter described.

Halogenation principally yields 6-halomethylene compounds and smallamounts of the 7 and/ or 9-halo compounds. Dihalogenation givessubstitution on the exo methylene and at predominately the 9-position.The coproduced isomers are separable from the principal products bystandard techniques, for example, fractional crystallization, columnchromatography and/or countercurrent distribution using known solventsystems.

The lla-dechlorination is accomplished by either chemical or catalyticreduction using procedures Well known to those in the art. Catalyticreduction may be accomplished in a solvent for the starting compound inthe presence of a noble metal catalyst and at pressures of hydrogen gasranging from atmospheric to superatmospheric. Temperatures of from 0 toabout 50 C., and usually room temperature, are preferred, and generallygive best results. The noble metal catalyst, e.g. palladium or rhodium,is advantageously employed on a support such as carbon, a commonlyavailable form. The hydrogenation is carried out until an equimolaramount of hydrogen gas is absorbed, at which point the hydrogenation isstopped. When the starting compound contains reducible groups other thanIla-halogen, e.g. 7 or 9-halogen, milder conditions of temperature andpressure should be used, particularly when palladium is used ascatalyst. The solvent selected for the hydrogenation should, of course,be reaction-inert, and a variety of organic solvents may be used forthis purpose; minimum laboratory experimentation will permit theselection of a suitable solvent for any specific starting compound.Generally, lower alkanols, e.g. methanol,

ethanol, are found most suitable, although a variety of other solventsmay be used.

A variety of chemical reducing agents may be used for the presentlla-dechlorination reaction. These include reduction with active metalssuch as zinc, or with active metals in mineral acids, e.g. zinc or ironin dilute hydrochloric acid; reduction with alkali metal hydrosulfite,preferably sodium hydrosulfite, which is commercially available, inaqueous media; and reaction with sodium iodide in a halogen-acceptorsolvent such as acetone or methanol, preferably in the presence of zincmetal. When aqueous systems are used in the aforementioned chemicalreductions, it is at times desirable-to utilize a water-misciblesolvent,particularly when the starting compound is of limited solubilityin the reaction mixture. The water-miscible solvent does not alter thecourse of the reduction, but merely provides for more efiicientreduction, e.g. shorter reaction time, by providing more intimatecontact of the reagents. A large number of such solvents are availableand include, among others, dimethylformamide, dimethoxyethane, methanol,ethanol, acetone, dioxane, tetrahydrofuran and the like. When thecompound to be lla-dechlorinated contains a nitro group, reduction withsodium iodide in acetone is a preferred method if the nitro group is tobe retained. On the other hand, if the corresponding 9-amino-lla-deshalo product is desired, catalytic hydrogenation, withcOnSuInptiOn of four equivalents of hydrogen, may be employed.

In general, deiluorination of the lla-fluoro derivatives by catalytichydrogenation is accompanied by prior or simultaneous reduction of theexocyclic double bond; these derivatives are therefore valuableintermediates for the preparation of 6-deoxytetracyclines. However,11adefluorination can be achieved with retention of the exocyclic doublebond, by chemical reduction with hydrosulfite or metal combinations, aspreviously discussed. 9-nitro groups, where present, are reduced toamino in these procedures.

The products are obtained from the lla-dehalogenation reaction mixturesby standard procedures. For example, the present new compounds areisolated from the catalytic hydrogenation reaction mixtures, afterfiltration of the catalyst, by precipitation, e.g. with a nonsolventsuch as ether or hexane, or concentration, usually under reducedpressure, or a combination of these. Work-up of the chemical reductionmixtures to obtain the reduction product may also. be accomplished byknown procedures, such as precipitation, concentration, solventextraction, e.g. with alcohols such as the butanols and pentanols, orcombinations of these procedures.

The reduction products, after isolation, may be purified by any of thegenerally known methods for purification of tetracycline. These includerecrystallization from various solvents and mixed solvent. systems,chromatographic techniques and counter-current distribution, all ofwhich are usually employed for this purpose.

The following table summarizes the activity of 6- methylenetetracyclinesagainst a variety of disease-causing microorganisms includingantibiotic-resistant strains. The minimum inhibitory concentration (MIC)is determined iby the well-known serial-dilution technique. Included inthe table are the MICs of the 11a-chloro-6- methylenetetracycline,6-methylenetetracycline and 6- deoxytetracycline. It is noted that theMIC values forv 6-methylenetetracycline are generally lower than thosefor 6-deoxytetracycline, indicating greater activity, es--- peciallyagainst Micrococcus pyogenes var. aureus 400, a tetracycline-resistantorganism.

TABLE I 1. fi-methylenetetracycline 2. fi-deoxytetracycline 3.11a-chloro-6-methylenetctracycline MIC (meg/ml.) Organism Micrococcuspyogmes var. caret/.8- 0.19 0. 78 (25 p)50 Streptococcus pyogenes. 0. 19O. 39 12. 5 Streptococcus jaecalis 0. 19 0. 78 25 Diplococcus pneilmoni0. l9 3. 12 25 Erysipelothrix rhasiopath 0. 19 0. 39 Carynebacteriumdiphtheriae O. 78 3. 12 100 Listeria monocytogenes O. 19 6. 25 25Bacillus subtilis 0. 19 0. O1 3. 12 Lactobacillas casei 0.78 25 (50 p)100 Bacterium ammoniagenes 0. 19 0. 78 5 Aerobacter aerogenes 3. 12 6. 3100 Escherichia coli- 1. 56 6. 3 100 Proteus vaZga-ris 12. 5 100 100Pseadomonas aerugiiio 25. 100 100 Salmonella galli'aaram 3. 12 12. 100Salmonella pullomm 0. 78 3. 12 (p) 100 Klebsiella pneumoniae- 1. 56 6. 3100 Neisseria gonorrhoeae-" 0. 1S) 0. 78 12. 5 Hemophilus influenzae- 0.19 0.09 6. Shigella sonnei 3. l2 3. 12 Brucella bronchiseptica O. 19 0.19 6. 25 Malleumyces mallet- 0. 39 3. 12 25 Vibrio comma 0.19 0.19 6. 25Pastearella multot'id O. 19 .0. 39 25 Streptococcus agalactiae" 0. 1912. 5 Mycobacterium 607 0. 19 0. 19 O. 78 Mycobacterium berolinense.0.19 100 0. 39 Candida albica-ns 50 100 100 Sarcina latca- 0. 19 100Antibiotic resistant strains of M z'crococcas pyogenes var. aureus:

1 Resistant to tetracycline at a concentration below 100 meg/mg. 2Resistant to tetracycline at a concentration below 50 meg/mg. (p)=partial inhibition.

When in vitro tests were repeated inthe presence of human serum, similarresults are observed. For example, -6-methylenetetracycline in 20% humanserum when tested against Micrococcus pyogenes var. aureus showed a MICvalue of 0.78; against Streptococcus pyogenes, 0.39. When1la-chloro-G-methylenetetracycline was tested in vivo through both theoral and parenteral routes in mice, it showed activity comparable totetracycline against infection produced with' tetracycline-sensitivemicroorganisms. The PD (PD=protective dose) for l1achloro-6-methylenetetracycline against an infection produced withMicrococcus pyogenes var. aureus is 5.5 mg./kg. (orally) and 1 rug/kg.(parenterally). Similar results are observed using6-methylehetetracycline. I

The present new 6-1nethylene compounds may be formulated into variouscompositions analogous to the parent tetracyclines from which they arederived. They are also useful therapeutically in feeds or as growthstimulants, in veterinary practice, and in agriculture. Prolonged oraland parenteral treatment of test animals with several of the new-6-methylenetetracyclines of the present invention has resulted inthyroid pigmentation.

Until these compounds have been clinically evaluated,

it is impossible to state whether the same manifestations will beassociated with prolonged treatment of man and Whether or not any suchmanifestations Will be reversible. In any case, these compounds willbeuseful for the oral or parenteral treatment of acute infections whichcan'be quickly cured, as well as for topical application purposes.

As will be apparent to those skilled in the art, the 6- methylenetetracyclines and the lla-halo-6-methylene tetracyclines may bepartially converted to their C epimers under many conditions,particularly at pI-Is between 2 and 6 and in such solvents as glacialacetic acid. In practice, 6-methylene tetracycline andllahalo-6-methylene tetracycline as isolated by the procedures describedherein may contain small amounts, i.e. less than 20%, of their Cepi-mers. 5-hydroxy-6- methylene tetracyclines are more resistant to Cepimerization. The C epimers of the compounds of this invention may beisolated from mixtures using standard procedures such as paperchromatography or countercurrent distribution techniques. Theessentially pure C epi-compounds .may be reconverted to the normal, moreactive form by procedures well known to the art, for exam le, bytreatment with glacial acetic acid.

Like the other new 6-methy1enetetracyclines of this invention, '6deoxy-6-dernethyl-6-nitromethylenetetracycline and the correspondingIla-halo compounds exhibit in vitro activity against a variety ofmicroorganisms. In addition, theyare useful intermediates for otherantimicrobial agents; under conditions which remove the Ila-halogen andreduce the nitro to an amino group, the resulting unstable 6deoxy-6-demethyi-G-arninomethylenetetracycline readily hydrolyzes toform 6-de oxy-6-demethyl-6-formyltetracycline, and this substance may befurther subjected to the numerous transformations characteristic of theformyl group, to produce additional products.

The new 9'-amino-6methyleneand -6-halomethylene tetracyclines, and thecorresponding lla-fluoro compounds, may also be converted to othervaluable antimicrobial agents, including9-formamido-6-methylenetetracycline, by standard acylation. The aminogroups are transformed, for example, to the corresponding 9- formamidoor acetamido derivatives by room temperature treatment with acylatingagents such as acetoformic anhydride or acetic anhydride, respectively.The same reactions may also be applied to the hereinafter describedderivatives of the 6-methylene and 6,-halomethylenetetracyclines,including the anhydrotetracyclines, dehydrotetracyclines andtetracyclines possessing a 9-an1ino group.

In addition to their use as antibacterial agents, the present newcompounds of Formulae I, II and III are useful for the syntheses of avariety of tetracyclines by conversion to correspondinga,6-anhydrotetracycline compounds, oxygenation of said compounds toproduce corresponding dehydro compounds, which, on reduction, areconverted to tetracycline compounds which differ from the starting6-methylene compounds by virtue of the hydroxy and other substituent atthe 6-position of the tetracycline nucleus.

The conversion to 5a,6-anhydrotetracyclines produces new compoundsrepresented by the formulae:

(XVII) in which X is selected from the group consisting of chloro, bromoand iodo; I X is selected from the group consisting of hydrogen andchloro;

X is selected from the group consisting of hydrogen,

chloro, bromo, iodo, nitro and amino; and X is selected from the groupconsisting of nitro and amino.

The conversion to anhydro compound is accomplished by treatment of thestarting 6-methylenetetracycline with an acid. A number of acids may beused for this purpose. Mineral acids are found to be particularlysuitable, for example, 85% aqueous sulfuric acid, or hydrochloric,hydrofluoric, phosphoric, perchloric acids, hydrogen chloride, hydrogenfluoride, and the like. Generally, it is sufficient to merely contactthe starting compound with the acid at from room temperature to about100 C. and merely allow the reaction to proceed to completion. Thecourse of the reaction may be readily followed by paper chromatographicanalysis of an aliquot of the reaction mixture, which allows fordetermination of the most efiicient reaction periods. The products areobtained by standard procedures of isolation, e.g. concentration andprecipitation. The new anhydrotetracyclines have substantialantibacterial activity in vitro. The 6-halomethyl anhydrotetracyclinesalso undergo standard nucleophilic displacement reactions with a varietyof nucleophils, including halides, nitrites, cyanates, thiocyanates,thiosulfates, amines, alkoxides and mercaptides, to yield otherantibacterial anhydrotetracyclines substituted at the 6-methyl group.

The oxygenation process to produce dehydrotetracyclines yields thefollowing new compounds:

no ornx T out)? -on on X CONH2 I II I OH OH O O (XVIII) X is selectedfrom the group consisting of hydrogen,

chloro, bromo, iodo and amino.

This process is accomplished by microbiological means. Theanhydrotetracycline selected is added to a fermentation mediuminoculated with. an S. aureofaciens strain such as S. aureofaciens ATCC12748, 12749, 12750, 12751 and 13191, which are obtainable from theAmerican Type Culture Collection, Washington, D.C. The fermentationmedium contains the usual nutrients and mineral substances used for thecultivation of strains of S. aureofaciens. The other general conditionsof the fermentation, such as pH, temperature, time, aeration, inoculum,sterilization and the like are conventional and may be similar to thosefor the production of chlortetracycline shown in US. Patent 2,482,055.

The product is obtained by conventional procedures; e.g. the broth maybe adjusted to pH 1-2 with mineral acid, filtered, and the filtrateadjusted to about pH 8-9 to precipitate the product. Alternatively, thefiltered broth may be extracted with solvent, such as b-utanol. Theextract is then concentrated and chromatographed on a diatomaceous earthcolumn in the Well known rnan ner and the column developed with :20butanol:chloroform. The efiiuent extracts are then concentrated andlyophilized. The crude product may then be crystallized from a suitablesolvent, e.g. methanol, if desired.

Alternatively, the conversion of the 5a,6-anhydrotetracyclines tocorresponding 5,5a dehydrotetracyclines may be effected by thephoto-oxidation procedure of Scott and Bedford, as described in theJournal of the American Chemical Society, volume 84, pp. 2271-2 (1962).In accordance with this procedure, the anhydrotetracycline is oxidizedto the corresponding 6-deoxy' -hydroperoxy dehydrotetracycline bycontacting a so-lution of the starting compound in a reaction-inertsolvent with oxygen or air while irradiating with light of about 300-450m wave length; and subsequently reducing the hydroperoxy compound to thedesired dehydrotetracycline, for example by treatment with an aqueoussolution of an alkali metal sulfite or hydrosulfite, or by hydrogenationin the presence of a noble metal catalyst such as palladium or rhodium.In the c-ase of catalytic hydrogenation, continued reaction may lead tofurther I -on OH" X5 CONH2 I Y on 0 (XIX) in which X is selected fromthe group consisting of chloro, bromo and iodo; and

X is selected :from the group consisting of hydrogen,

echloro, bromo, iodo and amino.

Various procedures, including catalytic hydrogenation andmicrobiological reduction, are effective for reduction. Themicrobiological reduction is effected by adding .respondingly greater.

the dehydrotetracycline to a fermentation medium inoculated with aconventional chlortetracyclineor tetracycline-synthesizing strain of S.aureofaciens, such as publicly available .5. aureofacz'ens NRRL 2209(obtainable from the Northern Regional Research Laboratories at Peoria,Illinois), and allowing fermentation to proceed under aerobicconditions. The conditions of the fermentation may be the same as theknown methods for producing chlortetracycline. and tetracycline byfermentation, except for the addition of one of the newdehydrotetracyclines at the beginning of, or during, the fermentation.The media and fermentation conditions set forth above for thepreparation of dehydrotetracyclines are also suitable here.

As an alternative to microbiological reduction, the conversion of the5,5a-dehydrctetracyclines of the present invention to the correspondingtetracyclines may be effected by catalytic hydrogenation. Thehydrogenation react-ion is carried out under conventional conditions.The dehydrotetracycline is dissolved in a reaction-inert solvent andthen subjected to treatment with hydrogen gas over a noble metalcatalyst, including palladium, platinu-m, rhodium, andthe like. Suitablesolvents include dimethyl formamide, dioxane, tetrahydrofuran,monoglyrne, diglyme, and the like. If desired, the catalyst may be onewhich is suspended in an' inert carrier, such as palladium on carbon.The hydrogenation may be carried out at atmospheric or superatmospherepressures of hydrogen gas, i.e. up to several thousand pounds per squareinch. It is generally preferred, however, to employ pressures of fromabout 2 to about. 4 atmospheres, since these are found most convenient.The reaction temperature does not appear to be critical. Excellentresults are obtained with temperatures up to about 50 C. The use ofhigher temperatures, though operable, is not recommended, since loweryields of the desired product may result.

I After the reaction is complete, as indicated by the absorption of onemole of hydrogen, the product is obtained in the usual manner, e.g.filtration of the catalyst and concentration of the reaction mixtures.The products may be further purified by countercurrent distribu tion inbutanol:0.0l N aqueous HCl. In the case of those dehydrotetracyclinescontaining a 7- or 9-halo group, hydrogenolysis of the latter mayconcurrently occur, in which case the hydrogen uptake will be cor Thelatter reaction is facilitated by the presence of a base, e.g.triethylamine. However, under mild conditions, and particularly whererhodium on carbon is employed as catalyst, it is possible to hydrogenateat the 5,5a-position without concurrent removal of halo substituents inthe D ring.

The new tetracycline compounds so produced are also useful for humantherapeutic uses, in agriculture and in veterinary practice.

For human therapy, the usual oral dosage of the present new tetracyclinecompounds described herein is from about 0.1 to about 2 g. per day forthe average adult. The products are formulated into capsules or tabletscontaining from 25 (0 250 mg. of antibiotic on an activity basis.Suspensions or solutions in various vehicles are prepared havingconcentrations ranging from to 125 mg./-rnl. For parenteraladministration intramuscularly or intravenously, the daily dose isreduced to about 0.2 to 1.0 g. Intramuscular formulations comprisesolutions of the antibiotic at concentrations ranging from 50 to 100rug/ml. Intravenous administration is by means of isotonic solutionshaving antibiotic concentration of about rug/ml. Both types ofparenteral products are conveniently distributed as solid compositionsfor reconstitution. In all instances, of course, the attending physicianwill indicate the dosage to fit the needs of a particular patient. Forchildren,v smaller doses are generally used.

The present invention embraces all salts, including acid-addition andmetal salts, of the newly recognized amphoteric antibiotics. Thewell-known procedures for preparing salts of tetracycline compounds areapplicable here and are illustrated by examples appearing hereinafter.Such salts may be formed with both pharmaceutically acceptable andpharmaceutically unacceptable acids and metals. Bypharmaceuticallyacceptable is meant those salt-forming acids and metalswhich do not substantially increase the toxicity of the amphotericantimicrobial agent. The preferred salts are the acid addition salts andpharmaceutically acceptable metal salts.

The pharmaceutically acceptable acidaddition salts are of particularvalue in therapy. These include salts of mineral acids such ashydrochloric, hydriodic,'hydrobromic, phosphoric, metaphosphoric, nitricand sulfuric acids, as well as salts of organic acids such as tartaric,acetic, citric, malic, benzoic, glycollic, gluconic, gulonic, succinic,arylsulfonic, e.g. p-toluene-sulfonic acids, and the like. Thepharmaceutically unacceptable acid addition 'salts, while not useful fortherapy, are valuable for isolation and purification of the newlyrecognized antibiotics. Further, they are useful for the preparations ofpharmaceutically acceptable salts. Of this group, the more common saltsinclude those formed with hydrofluoric and perchloric acids.Hydrofluoride salts are particularly useful for the preparation of thepharmaceutically acceptable salts, e.g. the hydrochloride, by solutionin hydrochloric acid and crystallization of the hydrochloride saltformed. The perchloric acid salts are useful for purification andcrystallization of the new anti biotics.

Whereas all metal salts may be prepared and are useful for variouspurposes, the pharmaceutically acceptable metal salts are particularlyvain-able because of their utility in therapy. The pharmaceuticallyacceptable metalsinclude more commonly sodium, potassium andallcalineearth metals of atomic number up to and including 20, i.e., magnesiumand calcium, and additionally aluminum, zinc, iron, and manganese, amongothers.-

Of course, the metal salts include complex salts, i.e., metal chelates,which are well recognized in the tetracycline art. The pharmaceuticallyunacceptable metal salts embrace most commonly salts of lithium and ofalkaline earth metals of atomic number greater than 20, i.e. barium andstrontium, which are useful for isolating and purifying the antibiotic.Since the new antibiotics are amphoteric, they also form salts withamines of sufiicient basicity.

It will be obvious that, in addition to their value in therapy, thepharmaceutically acceptable acid and metal salts are also useful inisolation and purification of the present new compounds. i

The following examples are given by way of illustration and are not tobe construed as limitations of this invention, many variations of whichare possible within the scope and spirit thereof.-

Example Z.-11a-chloro-6-deoxy-6-demethyI-6- methylenetetracycline 'isdissolved in 350 ml. of H 0 by warming and stirring.

An equal volume of cone. HCl is added to the clear solution and theproduct crystallizes as the hydrochloride salt. Elemental analysis ofthe hydrochloride salt thus obtained gives the following results.

Calcd. for C H O N Cl C, 53.'ll; H, 4.56; chloride 7.13; N, 5.63. Found:C, 52.62; H, 4.63; chloride 6.84; N, 5.54.

Infrared analysis of the product as the hydrochloride salt in a KBrpellet at 1% concentration shows carbonyl absorption at 5.70 as well asthe following significant peaks: 6.1, 6.23, 6.36, 6.45 (shoulder), 6.91,7.85, 8.14, 8.55, 10.22, 10.55 and 10.89. Bioassay of the product (K.pneumoniae) shows an oxytetracycline activity of 50l00 meg/mg. Ultraviolet analysis of the sample in 0.01 N methanol-HCl shows maxima at376, 278 and 242 me The product shows an Rf value of from 0.2 to 0.3 inthe following system.

Mobile phase: 20:3 toluene-pyridine saturated with pH 4.2 bufferImmobile phase: pH 4.2 buffer (aqueous) On analysis of the papergramwith ultraviolet light, the product spot does not fluoresce strongly.However, on spraying, with aqueous sodium hydrosulfite, it shows strongfluorescence.

The C epimer of the product, present as a minor impurity, shows R 0.1 inthis system.

Example 2.-1Za-flu0r0-6-de0xy-6-demethyl-6- methyl enetetracycline1la-fluorotetracycline-6,l2-hemi-ketal, 250 mg. is stirred in 2 ml. of63% aqueous perchloric acid. The solid dissolves on warming to 6065 C.for 15 minutes after which the mixture is cooled and water is added toobtain l1a-fluoro-6-deoxy-6-dernethyl-6-me-thylenetetracycline as thechlorate salt. The product shows similar absorption on ultravioletanalysis to that of Example 1.

Example 3 The following compounds are prepared from corresponding11a-halotetracycline-6,IZ-hemi-ketals by the procedure of Example 1:

7,1la-dichloro-6-deoxy-6-demethyl 6 methylenetetracycline 7-bromo 11achloro 6 deoxy 6 demethyl 6- methylenetetracycline Example 4.Preparatinof mineral acid salts lla-chloro 6 deoxy 6 demethyl 6methylenetetratetracycline hydrofluoride is dissolved in water and thesolution adjusted to pH 5. The resulting amphoteric compound whichprecipitates is filtered and dried. The amphoteric compound is dissolvedin methanol containing a molar equivalent of hydrogen chloride and thehydrochloride salt is precipitated by the addition of ether.

This hydrochloride salt is also prepared by the method of Example 1 fromthe hydrofluoride salt. Other mineral acids may be substituted forhydrochloric acid in this procedure to obtain the salt of choice.

The hydrochloride salt is recrystallized from butanol,butanol-hydrochloric acid, acetone or acetone-hydrochloric acid. Thehydrofluoride salt used as starting compound may be recrystallized frommethanol, methanol-monoglyme, or methanol and ethylene dichloride.

Using similar procedures, the hydrochloride, hydrobromide, nitrate,sulfate, hydriodide and phosphate salts of the Ila-halo 6methylenetetracyclines of the previous examples are prepared.

Similarly, other salts are formed using a variety of acids, for example,organic carboxylic acids such as tartaric, citric, malic, benzoic,glycollic, gluconic, gulonic, succinic, acetic and the like. The saltsformed with pharmaceutically-acceptable acids are useful for therapy;those with pharmaceutically-unacceptable acids are useful in thepurification of the new products and in the preparation ofpharmaceutically acceptable salts.

Example .6-deoxy-6-demethyZ-6-methylcnetetracycline METHOD A The productof Example 1 (5 mg.) is dissolved in 3 ml. of methanol and a freshlyprepared solution of sodium hydrosulfite (20 mg. in 2 ml. of water) isadded. The mixture is allowed to stand for 15 minutes at room ternperature, after which it is stripped of methanol and extracted withbutanol. The butanol extract is concentrated to obtain the product, 6deoxy 6 demethyl- 6-methylene tetracycline. The product is crystallizedfrom water as the p-toluene-sulfonate salt by addition ofp-toluenesulfonic acid. Alternatively, it is crystallized as thehydrochloride salt from water by addition of concentrated HCl.

In a repeat experiment on a larger scale, 500 mg. sodium hydrosulfite iscombined With one gram of the substrate in 25 ml. water plus 25 ml.methanol. The reaction mixture is stirred 30 minutes at room temperatureand then clarified by filtration. The methanol is evaporated from thefiltrate and the residue is extracted with,

butanol. product.

On testing against K. pneumoniae, the product has an oxytetracyclineactivity of at least 1100 meg/mg. The product shows an R value of 0.6 inthe same system as described in Example 1, and shows Rf 0.9 in thefollowing system.

Evaporation of the extract yields the desired Mobile phase: 20: 10:3nitromethane:chloroforrn:pyridine Immobile phase: PH 3115 buffer(aqueous) The C epimer of this substance shows Rfs ofapproximately 0.4and approximately 0.6 in the two systems.

METHOD B A solution of g. of 11a-chloro-6-deoxy-6-demethyl- 6methylene-tetracycline hydrochloride in 500 ml. of monomethyl ether ofethylene glycol is cooled to 4 C. in an ice bath and the rapidly stirredsolution is treated with 50 g. of zinc metal dust at a gradual rate overa period of about 10 minutes. The temperature rises to- 12 C. duringaddition. After addition is complete, the temperature begins to fall.After a total reaction time of 15 minutes the zinc is removed by rapidfiltration and Washed with solvent. One liter of Water is addedgradually to the filtrate over a period of about 10 minutes. A yellowslurry of the zinc complex of the product is formed. pH of the solutionis then adjusted to 6.8 with 10% aqueous sodium hydroxide.

The resulting slurry is digested for about 1.5 hours in an ice bath andfiltered. The Wet cake is then slurried in 750 ml. of water and cone.HCl is added dropwise until a clear solution is obtained. A slightexcess of cone. HCl causes rapid crystallization of 6-deoxy 6 demethyl-6 methylenetetracycline hydrochloride as glistening needles. Afterdigestion for one hour, the product is filtered and dried. The yield ofproduct is 37.8 g. The product melts at 213.8-214.2 C. withdecomposition.

, METHOD 0 11a fluoro 6 deoxy-6-demethyl-6-methylenetetracycline, 5 g.in 125 ml. dilute hydrochloric acid (1 part cone. HCl in parts water),is combined with 2 g. zinc dust and stirred one hour at 20 C. Thereaction mixture is then filtered and the filtrate extracted withbutanol. Evaporation of the butanol extract yields the desired productcontaining some anhydro compound as impurity.

METHOD D To one gram of the same starting compound as in Method C in 25ml. water plus 25 ml. methanol is added 400 mg. sodium hydrosulfite.Stirring is continued for 20 minutes at room temperature. An additional400 mg. sodium hydrosulfite is then added and stirring continued 20minutes longer. This step is repeated a second time and the reactionmixture is finally clarified by filtration and stripped of methanol.Butanol extraction of the residue and evaporation of the extract yieldsthe product.

Example 6 The procedure of Example 5, Method A, is repeated todechlorinate the 11a-chloro-6-methylenetetracyclines of the previousexamples to provide the following products:

7-bromo-6-deoxy-6-demethyl-6-methylenetetracycline7-chloro-6-deoxy-6-demethyl-6-methylenetetracycline C epimers occur asimpurities.

Example 7 The hydrochloride, hydrooromide, sulfates, hydriodide andphosphate salts of the G-methylenetetracyclines are prepared accordingto the methods described in Example 4.

Example 8 Example9.11a-chloro-6-a'e0xy-6-demeflzyl-d-chloromethylerzetelracycline METHODA To a mixture of 1.2- g. of the Example 1 product in 15 ml. oftrifluoroacetic acid is added 350 mg. of N-chlorosuccinimide and themixture is heated at 60 C. After about 3 hours, the mixture gives anegative KI/starch test. The cooled mixture is added dropwise to 500 ml.of cold ether with stirring at ice-bath temperature. After stirring for3 hours, the precipitated product is filtered, twice slurried in etherand dried to obtain 1 g. of product. The product is dissolved in 300 ml.of hot methanol, filtered to removea small amount of insoluble materialand concentrated to a volume of 100 ml. Then 3 ml. of p-toluenesulfonicacid dissolved in methanol is added and the mixture permitted to standatroom temperature until crystallization is completed. The product isobtained as the p-toluenesulfonate salt by filtering the precipitate,Washing with methanol and drying.

Infrared analysis of the product shows a clear shar curve with a band at5.69;.

Ultraviolet analysis in 0.01 N methanolic HCl shows maxima at 245 and378 m Elemental analysis gives the following results:

Calcd. for C H O N Cl S (as the p-toluenesulfonate salt): C, 52.18; H,4.2; N, 4.2; Cl, 10.6; S, 4.8. Found: C, 51.8;1-1, 4.3; N, 4.3; Cl,10.3; S, 4.8.

The PD in mice via the parenteral route vs. Staph. aureus infection is20 mg./ kg.

METHOD B 1 gram of the product of Example 1 is placed in a polyethylenecontainer maintained in an ice Water bath. To this is added 5 ml. ofliquid hydrogen fluoride, followed by 330 mg. of N-chlorosuccinimide.The mixture is allowed to stir at ice bath temperature for one hour, andthen poured into ml. of methanol containing 1.2 g. paratoluene sulfonicacid. Ether, 25 ml., is slowly added, and the product crystallizes asthe pure toluenesulfonate salt. It is recovered by filtration, washedwith ether and dried, to yield 950 mg. Alternatively, the HF reactionmixture is poured into isopropanol containing nitric acid and theproduct isolated as the nitrate salt.

Following the procedures described, 1la-fluoro-6-deoxy-6-demethyl-6-methylenetetracycline is converted to thecorresponding 11a-fluoro-chloro-methylene compound.

To a mixture of 4.8 g. of the Example 1 product in 40 ml. oftrifluoroacetic acid is added a solution of 0.54 ml. otbromine in 10 ml.of acetic acid. The mixture is warmed at 40 60 C. for one hour and thenallowed'to stand for 12 hours at room temperature. The crude product isobtained by the same Work-up as in the previous example. It iscrystallized from methanol as the ptoluenesulfate salt (3.54 g.).Ultraviolet analysis in 0.01 N methanolic HCl shows maxima at 249 and379 mp.

In the same way, 11a-fluor0-6-deoxy-6-demethyl-6- methylenetetracyclineis converted to the corresponding lla-fluoro-bromomethylene compound.

Example 11 .I1a-chloro9-bromm6-de0xyo-dembthyld-bromomethylenete'tracycline To a mixture of280 mg. of the Example 1 product in 5 ml. of trifiuoroacetic acid isadded 1.05 ml. of a solution of 0.53 ml. of bromine in 10 ml. of aceticacid. A

heavy orange precipitate forms in the mixture, which is then stirred andwarmed at 60 C. for 2 hours. The mixture is then allowed to stand for 48hours and the product (287 mg.) then obtained by work-up with ether aspreviously described. The product is crystallized as the sulfate salt bydissolving in 5 ml. of methanol and adding 6 drops of conc. H 50Ultraviolet analysis of the product in 0.01 N methanolic HCl showsmaxima at 251 and 384 mu.

Example 12.-9,11a-dichlor0 6-deoxy-6-demwhy [-6- chlorom ethylenetetracycline Thisproduct is prepared according to the procedure ofExample 9 by using two molar equivalents of N-chlorosuccinimide.

Example 13 .--1Ia-chloro Q-nitr0-6-de0xy-6-demethyl-6-methylenetetmcwcline METHOD A A mixture of 1 g. of the Example 1product in 20 ml. of 50% aqueous acetic acid containing 1 ml. of conc.HNO is allowed to stand for 12 hours, as the product crystallizes as thenitrate salt. The product is collected by filtration, water-washed anddried.

METHOD B 11a chloro 6 demethyl-6-deoxy-6-inethylenetetracyclinehydrochloride, one gram, is suspended with stirring in 40 ml. 5% aqueousnitric acid at 60 C. After 24 hours, an orange solid is recovered byfiltration. The wet cake is taken up in 300 ml. hot methanol, treatedwith activated carbon, filtered and the filtrate concentrated to 20 ml.5% nitric acid, about 1 ml., is added, and the product allowed tocrystallize as the nitrate salt on overnight refrigeration. Yield is mg.

The products of these procedures exhibit ultraviolet absorption maximain methanol: 0.01 N HCl at 257 and 37 me; and in methanol: 0.01 N NaOHat 254 and 460 mu.

The following additional nitro compounds are prepared from thecorresponding products having hydrogen at the 9 position by theforegoing methods:

7,1 1a-dichloro-9-nitro-6-demethyl 6-deoxy-6-methylenetetracycline 11a-chloro-9-nitro-6-demethyl-6-deoxy-6-ch1orometh ylenetetracycline I 11a-chloro-9-nitro-6-demethyl-6-deoxy-6-bromomethylenetetracycline l1a-chloro-7-bromo 9-nitro- 6-deoxy-6-demethyl-6-methylenetetracycline 11a-fluoro-9-nitro-6-deoxy-6-demethyl-6-methylenetetracycline l1a-fiuoro-9-nitro 6-demethyl-6-deoxy--chloromethlyenetetracycline '1 7 11a-fluoro 9-nitro-6-demethyl-6-deoxy-6-bromomethylenetetracyclineExample 14.-11a-chlor0-6-de0xy-6-demethyl-6-nitr0- methylenetetracyclineOne gram of the Example 1 product is stirred in 5 ml. trifiuoroaceticacid containing 0.15 ml. 70% nitric acid. The resulting solution isallowed to stand for about 24 hours and then poured into 200 ml. etherto precipitate about 1.1 g. of the desired product, which is collectedby filtration and dried.

Example 15.] 1a chloro-9mitro d-deoxy-6-demefhyl-6-chloromethylen'eletracycllne This product is obtained from that ofExample 13 by the chlorination procedure of Example 9, Method A.

Example l6.6 deoxy-6-demethyl-6-chl0r0methylenetelracycline METHOD A To1 mmole of the Example 9 product in 25 ml. of methanol is added 100 mg.of 5% rhodium on carbon. The mixture is hydrogenated with shaking atroom temperature and one atmosphere of hydrogen gas until 1 mmole ofhydrogen is taken up. After filtering off the catalyst, the solution isevaporated to dryness under reduced pressure. The residue is slurried inether, filtered and dried to obtain the product.

METHOD B The product of Example 9, 6.7 g. as the p-toluene sulfonatesalt, is stirred in 400 ml. of 50% methanol-water at room temperature.1.34 g. of sodium hydrosulfite is added to the slurry, and stirring iscontinued for 45 minutes. A second 1.34 g. portion of sodiumhydrosulfite is then added, and stirring continued for another 45minutes. Insoluble material (0.57 g.) is removed by filtration.

The filtrate is stripped under vacuum to remove the methanol, leaving ayellow-orange slurry. The slurry is extracted with several portions ofbutanol, and the hutanol extracts are combined and evaporated to drynessunder vacuum to yield the crude product. Ether is added, and the mixtureis stirred and then filtered to obtain 3.4 g. of final product.

Using these procedures, the following compounds are prepared from thecorresponding 11a-halo compounds:

6-deoxy-6-demethyl-6-bromomethylenetetracycline9-bromo-6-deoxy-6-demethyl-6-bromomethylenetetracycline9-chloro-6-deoxy-6-demethyl-6-chloromethylenetetracycline6-deoxy-6-demethyl-6-iodomethylenetetracycline7-chloro-6-methylenetetracycline 7-bromo-6-methylenetetracycline9-bromo-6-demethyl-6-deoxy-6-chloromethy1enetetracycline Example l7.9nitr-6-d'emethyl-6-de0xy-6-methylenetetracycline 11a chloro 9 nitro 6demethyl 6 deoxy 6- methylenetetracycline, g. as the nitrate salt(prepared as in Example 13), is stirred with 10 g. sodium iodide in 200ml. acetone for about 5 minutes, and then filtered to clarify. Theproduct precipitates from the filtrate upon standing overnight and isrecovered by filtration and dried. 4.0 g. of product are obtained,containing some of the corresponding 521,6-anhydro compound as a minorimpurity. An ultraviolet absorption maximum is observed at about 368 my.

Application of the foregoing procedure to the other 11a-chloro productsof Example 13 produces the following compounds: 1

7-chloro-9-nitro-6-demethyl-6-deoxy-G-methyIenetetracycline9-nitro-6-demethyl-6-deoxy-6-chloromethylenetetracycline 49-nitro-6-demethyl-6-deoxy-6-bromomethylenetetracycline 7-bromo-9-nitro-6-deoxy-6-demethyl-6-methylenetetracycline6-deoxy-6-demethyl-6-nitromcthylenetetracycline is prepared by treatmentof the corresponding 11a-chloro compound overnight at room temperaturewith sodium iodide in acetone in the proportions given above. Thereaction mixture is then evaporated to dryness under vacuum, the residuedistributed between butanol and water, and the butanol layer evaporatedto provide the product.

Example 18.9-amin0-6-a'e0xy-6-demethyl-6-methylenetetracycline METHOD ATo 1 mmole of the Example 13 product in 25 ml. of methanol is added mg.of 5% rhodium on carbon. The mixture is hydrogenated with shaking atroom temperature and one atmosphere of hydrogen gas until 4 equivalentsof hydrogen are taken up. After filtering off the catalyst, the solutionis evaporated to dryness under reduced pressure. The residue is slurriedin ether, filtered and dried to obtain the product.

The same product is obtained from 9-nitro-6-methylenetetracycline atcorrespondingly lower hydrogen uptake.

Using this procedure, the following compounds are prepared from thecorresponding 11a-chloro compounds of Example 13.

7-chloro-9-amino-6-demethyl-6-deoxy-6-methy1enetetracycline,9-amino-6-demethyl-6-deoxy-fi-chloromethylenetetracycline9-amino-6-demethyl-6-deoxy-6-bromomethylenetetracycline7-bromo-9-amino-6-deoxy-6-demethyl-6-methylenetetracycline METHOD B Thetitle product is also obtained from 9-nitro-6-methylenetetracycline andthe corresponding 11a-chloro and l la-fluoro derivatives by thehydrosulfite procedure of Example 5, Method D.

METHOD C The title product is also obtained from l1a-fluoro-9-nitro-6-methylenetetracycline by the zinc reduction of Example 5, MethodC.

Example 19.9 amino 6 de0xy-6-demethyl-6-chl0v'0- methylenetetra'eyclineMETHOD A 11a fluoro-9-nitro-6-chloromethylenetetracycline, 1 g., iscombined with 15 ml. 5% hydrochloric acid and 4.2 equivalents stannouschloride. After stirring for one hour at room temperature, the reactionmixture is butanolextracted and the product recovered by evaporation ofthe extract. The same product is obtained from the corresponding11a-chloro starting compound by this procedure.

METHOD B The same starting compound yields the title product by thehydrosulfite procedure of Example 5, Method D.

METHOD C The same starting compound yields the title product by the zincreduction of Example 5, Method C, with the substitution of 1:7 conc.HClzWater for the 1:55 acid of that earlier example.

9 amino 6 deoxy 6 demethyl 6 bromomethylenetetracycline is also obtainedby these procedures from the corresponding starting compound.

- 19 Example 20.-6 demethyl-6-chloromethyl-5a,6-anhydrotetracyclineMETHOD A Three grams of the Example 16 product is dissolved in 15ml. ofdry, liquid hydrogen fluoride and the mixture heated for 48 hours at 75C. in a stainless steel bomb. The mixture'is then evaporated to drynessunder nitrogen and the residue triturated with ether, filtered, washedwith ether and dried.

METHOD B 1 g. of the same starting compound as in Method A is heated atreflux temperature for 24 hours in a mixture of 10 ml. acetone and 10ml. dimethylformamide. The reaction mixture is then taken to dryness ona rotating evaporator under high vacuum to obtain the product.

METHOD C A mixture of 1.5 g. of the same starting compound in 25 ml.hydrochloric acid is combined with 3 ml. of a solution of 12 g. iodineand 8.5 g. potassium iodide in 25 ml. water. The reaction mixture isstirred overnight at room temperature, and an equivalent proportion ofsodium thiosulfate is then added to destroy the iodine. The product isrecovered by butanol extraction followed by evaporation of the combinedextracts under vacuum.

METHOD D 1 g. of the same starting compound in 20 ml. 20% hydrochloricacid is subjected to one atmosphere hydrogen pressure for 2 hours atroom temperature in the presence of 200 mg. 5% palladium on carbon. Thereaction mixture is then filtered and the filtrate diluted withlwater.The product is recovered from the diluted filtrate by butanolextraction, followed by evaporation of the combined extracts undervacuum.

Using these procedures, the following compounds are prepared fromcorresponding starting compounds:

6-demethyl-6-bromomethyl-5a-,6-anhydrotetracycline9-bromo-6-demethyl-6-bromomethyl-5a,6- anhydrotetracycline 9chloro-6-demethyl 6-chloromethyl-S 21,6-

,anhydrotetr-acycline 9-nitro-5a,6-anhydrotetracycline9-nitro-6-demethyl-6-chloromethyl-5 a,6-

anhydrotetracycline 9-nitro-6-demethyl-6-bromomethyl-5a,6-

anhydrotetracycline 9-amino-5a,6-anhydrotetracycline9-amino-6-demethyl-6-chloromethyl-5a,6-

anhydrotetracycline 9-amino-6 demethyl-6-bromomethyl-5 a,6-

anhydrotetracycline' 7 -chloro-'9-amino-5a,-6-anhydrotetracycline7-chloro-9-nitro-5a,6-anhydrotetracycline The above enumerated aminocompounds are also prepared from the corresponding nitro anhydrocompounds by reduction, e.g. by catalytic reduction over rhodium by themethod previously described.

9-nitro-5ag6-anhydrotetracycline is also prepared from9-nittro-6-demethyl-6-deoxy-G-methylenetetracyoline in the followingmanner: 500 mg. of the product of Example 17 is dissolved in 20 ml.methanol by warming, and 600 mg. 2-naphthalene sulfonic acid is added.The reaction mixture is permitted to stand at room temperature for 60hours and then is clarified by filtration. "10 ml. isopropan'ol is nextadded, and the mixture is heated to boiling for 5 minutes. 200 mg. ofthe desired product in the form of the naphthalene sulfonate saltcrystallizes from the reaction mixture upon cooling to room temperature,and an additional 190 mg. is obtained from the mother liquor in twosucceeding crops.

20 Example 2I.6-demeth yl-6-chloromezhyl- 5,5a-dehydrotetracyclineMETHOD A A sterile water suspension containing 60-80 million spores perml. of S. aureofaciens' ATCC 12,748 is employed to inoculate 24 volumesof a sterile tap water medium containing the following ingredients perliter.

Sucrose g Ammonium sulfate g 2 Calcium carbonate g 7 Cornsteep liquor ml16.5

The inoculated medium is incubated for 24 hours at 28 C. with shaking.

This inoculum is introduced into 25 times its volume of sterile watermedium containing the following ingredients per liter.

. I Grams Ammonium sulfate 5 Calcium carbonate 9 Ammonium chloride 1.5Magnesium chloride hexahydrate 2 Ferrous sulfate heptahydrate 12Manganese sulfate tetrahydrate 10 Cobalt chloride hexahydrate 1 Zincsulfate heptahydrate 20 I Cornsteep liquor 25 Starch 55 The inoculatedmedium is incubated at 25 C. on a rotary shaker for 48 hours. Atthispoint, 6-deoxy-6-demethyl-6-chloromethyl-5a, 6-anhydrotetracyclineis added to the broth under sterile conditions at a concentration of 0.5-g./l. and the fermentation is allowed to proceed for an additional 72hours under aerating conditions.

The fermentation is then halted and the product recovered by adjustingthe pH of the broth to 1.5 with cone.

HCl and filtering. The filter cake is washed with dilute acid, thecombined filtrate and washings are treated with sodium chloride (54 g.per 200 ml. of filtrate) and extr-acted with n-butanol. The combinedbutanol extracts are concentrated .to about one-tenth of the originalvolume, saturated with water and filtered. The filtrate is passed onto acolumn of diatomaceous earth (Johns- Manville Celite) containing 0.5 ml.of water (adjusted to pH 2 with HCl) per gram of Celite. The column isthen developed with butanol-ohloroform saturated with 0.01 N HCl toobtain the product in the major fractions which are concentrated toobtain the product.

METHOD B A 0.1% w./v. solution in toluene of the anhydrotet racyclinestarting compound of Method A is exposed to light of 300-450 mg wavelength at room temperature 7 while oxygen is introduced beneath thesurface at a rate 6-demethyl-dbromomethyl-S ,5 a-dehydrotetracycline9-bromo-6-demethyl-6-bromomethyl-5,5a-

dehydro tetracycline 9-chloro-6-demethyl 6-chloromethyl-5,5a-

dehydrotetracycline 9-amino-5 ,5 a-dehydrotetracycline 21 9 -amino-6demethyl-6chlorornethyl-5 ,S-a-dehydrotetmacycline9-amino-6-dernetl1yl-6=bromome-thyl-5,5a-dehydrotetracycline Example22.6-demethyl-6-chloromethyltetracycline A sterile aqueous fermentationmedium containing the following ingredients per liter is inoculated withS. aureofaciens NRRL 2209:

Calcium carbonate -g 9 Ammonium sulfate g 5.6 Manganese sulfate mg 80Cobalt chloride hexahydrate mg Ammonium chloride g 1.7 Starch g 55Cornsteep liquor g 25 Lard oil ml 32 To this mixture is added6-demethyl-6-deoxy-G-chloromethyl-5,Sa-dehydotetracycline .at aconcentration of 0.5 g./l. and fermentation is carried out at 25 C. witha-gitation for 120 hours. The product is obtained by sepa-. ration andpurification using standard procedures, e.g. as previously described.

Using this procedure, the following compounds are prepared fromcorresponding 5,5a-dehydro compoundsz'6-demethyl-6-bromomethyltetracycline9-bromo-6-demethyl-6-bromome-thyltetracycline9-chloro-6-demethyl-6-chloromethyltetracycline 9-aminotetracycline9-amino-6-demethyl-6-chloromethyltetracycline9-amino-6-demethyl-6-bromomethyltetracycline Example 23.Catalyticreduction of dehyro compounds6-demethyl-6-chlorornethy'l-5,5a-dehydrotetracycline is hydrogenated indimethylformamide over 5% rhodium on carbon at room temperature and 50p.s.i. hydrogen gas until 1 molar equivalent of hydrogen is taken up.The product, 6-demethyl-6-chloromethyltetracycline is obtained byrfiltering off the catalyst and concentrating the reaction mixture.

Using these procedures the remaining products of the previous exampleare obtained from corresponding dehydro compounds.

In those cases where halogen is present, the reduction is permitted tocontinue until a further equivalent amount of hydrogen is taken up toobtain the corresponding deshalo compound. This catalytic hydrogenationprocedure is repeated using palladium on carbon as the catalyst withsimilar results.

Example 24.-Acid addition salts Example 25.Metal salts The sodium saltof 6-demethyl-6-chlorome-thyltetracycline is prepared by dissolving theamphoteric substance in water containing an equimolar amount of sodiumhydroxide and freeze drying the resulting mixture.

In this fashion, other metal salts are prepared including potassium,calcium, barium, lithium and strontium salts.

The metal salt complexes of the present new tetracyclines are preparedby dissolving them in a lower ali- 22 phatic alcohol, preferablymethanol, and treating with a equimolar amount of the selected metalsalt, preferably dissolved in the selected alcohol. The complexes areisolated in some instances by simple filtration, but often, since manyof them are alcohol soluble, by evaporation of the solvent or additionof a non-solvent such as diethyl ether.

In this fashion, metal salt complexes of the present new tetracyclinesconsisting primarily of compounds containing a 1:1 ratio of metal totetracycline are prepared employing the following metal salts: calciumchloride, cobalt chloride, magnesium sulfate, magnesium chloride,stannous chloride, zinc chloride, cadmium chloride, barium chloride,silver nitrate, stannous nitrate, strontium nitrate, magnesium acetate,manganous acetate, palladium chloride, manganous chloride, ceriumchloride, titanium chloride, platinum chloride, vanadium chloride,plumbous acetate, stannous bromide, zinc sulfate, chromous chloride andnickellous chloride.

Example 26 A suspension of 6-demethyl-6-chloromethyltetracycline isprepared with the following composition:

Antibiotic g 31.42 70% aqueous sorbitol g 714.29 Glycerine, U.S.P. g187.35 Gum acacia (10% solution) ml Polyvinyl pyrrolidone g 0.5 Butylparahydroxybenzoate (preservative) g 0.172 Propyl parahydroxybenzoate(preservative) g 0.094

Water, distilled, to make 1 liter.

To this suspension, various sweetening and flavoring agents, as well asacceptable colors, may be added by choice. The suspension containsapproximately 25 mg. of antibiotic activity per milliliter.

Example 27 A solution of 6-demethyl-6-chlorornethyltetracycline isprepared with the following compositions:

Antibiotic g 30.22 Magnesium chloride hexahydrate g 12.36Monoethanolamine ml 8.85 Propylene glycol g 376 Water ml 94 The solutionhas a concentration of 50 mg./ml. and is suitable for parenteral andespecially for intramuscular administration.

1 Example 28 A tablet base is prepared by blending the followingingredients in the proportion by weight indicated: Sucrose, U.S.P. 80.3Tapicoa starch 13.2 Magnesium stearate 6.5 Into this base there isblended sufiicient 6-dernethyl-6-chloromethyltetracycline to providetablets containing 25, 100 and 250 mg. of active ingredient.

Example 29 A blend is prepared containing the following ingredients:

Calcium carbonate, U.S.P. 17.6 Dicalcium phosphate 18.8 Magnesiumtrisilicate, U.S.P. 5.2 Lactone, U.S.P. 5.2 Potato starch 5.2 Magnesiumstearate A 0.8

Magnesium stearate B 0.35 To this blend is added sufiicient6-demethyl-6-chloromethyltetracycline to provide capsules containing 25,100 and 250 mg. of active ingredient.

Example 30 One thousand grams of 6-demethyl-6-chloromethyltetracyclineare intimately mixed and ground with 2500 grams of ascorbic acid. Theground, dry mixture is filled into vials, sterilized with ethylene oxideand the vials sterilely stoppered. For intravenous administrationsuflicient water is' added to the vials to form a solution containing 10mg. of active ingredient per milliliter.

Example 31 .-11a-fluorotetracycline-6JZ-hemi-ketal To a suspension of 20g. of tetracycline base in 800 ml. of water cooled to C. is added 45 ml.(2 equivalents) of 2 N sodium hydroxide solution. The tetracyclinedissolves to yield a solution of pH approximately 11. Perchlorylfluoride is then bubbled through the stirredsolution (maintained under anitrogen atmosphere) until the pH of the mixture approximates 7. A heavyprecipitate starts to form between pH 8 and 8.5. The excess perchlorylfluoride is flushed out with a stream of nitrogen I Example32.11a-chloroteiracycline-tSJZ-hemi-ketal To a.solution of 2.2 g. ofanhydrous tetracycline in 25 ml. of monoglyme (dimethyl ether ofethylene glycol) is added. 800 mg. of N-chlorosuccinimide with stirringto dissolve the reagent. The mixture is allowed to stand for sevenminutes and then diluted with water (25 ml.). The product, 873 mg,crystallizes as white needles. Bioassay of the product shows atetracycline activity of about 4 meg/mg. against K. pneumoniae. Infraredanalysis shows no carbonyl bands between 5 and 6 microns. Ultravioletabsorption shows maxima at 267 and 340-342 m On treatment of thisproduct with sodium hydrosulfite in aqueous dimethylformamide at roomtemperature, tetra-cycline is regenerated. Bioassay of the reactionmixture shows a tetracycline activity of 520665 meg/mg. (K. pneumoniae).

The crystalline hydrochloride of this product is obtained by dissolvingit in excess aqueous HCl (pH ca. 1) and freeze drying the mixture. 7

Example 3 3 7 ,1 I a-dichZorotetracycline-(SJ Z-hemi-ke tal A mixture of2.4 g. of anhydrous 7-chlorotetracycline, 800 mg.'of-N-chlorosuccinimideand 25 ml. of the dimethyl ether of ethylene glycol is stirred for 2 /2minutes after which 100 ml. of ether is added followed by 300 ml. ofhexane. The precipitate thus formed is collected by filtration, washedwith hexane and dried.

In similar fashion, 7-bromo-1la-ehlorotetracycline- 6,12-hemi-ketal isprepared from 7-bromotetracycline.

What is claimed is:

1. A compound selected from the group consisting of compounds of theformulae:

CHXi N(CH3)2 OH on]? .\/CONHz i (in (i 11 0 qHX N(CH3)Z /K OH at X;CONH5 be ti )11 d 24 in which X is selected from the group consisting ofchloro,

bromo and iodo; X is selected from the group consisting of chloro,bromo, iodo and nitro; and X is selected from the group consisting ofchloro,

bromo, iodo, nitro and amino; and acid addition salts andpharmaceutically acceptable metal salts thereof.

2. fi-deoxy-6-demethyl-6-chloromethylenetetracyline. 3.6-deoxy-6-dernethyl-6-nitromethylenetetracyline. 4.6-deoxy-6-demethyl-6-bromomethylenetetracyline. 5.6-deoxy-6-demethyl-6-chloromethylene-9-nitrotetracyclinc.

6. 6-deoxy-6-dernethyl-6-chloromethylene-9-aminotetracycline.

7. A compound selected from the group consisting of compounds of theformulae:

( 13x mom):

0H on V CONH2 Y (in ii 5 d 011:: mom

X1 GONH2 l \l n t OH O O O in which X is selected from the groupconsisting of chloro, bromo and iodo; X is selected from the groupconsisting of chloro,

bromo, iodo and nitro; and Y is selected from the group consisting ofchloro and fluoro; and acid addition salts and pharmaceuticallyacceptable metal salts thereof.

8. 11a-chloro-6-deoxy-6-demethyl-6-chloromethylenetetracycline. v 9.11a-chloro-6-deoxy-6-demethyl-6-nitromethylenetetracycline.

10. A compound selected from the group consisting of compounds of theformula:

cnxi 0111):

I H I II E O OH 0 wherein X is selected from the group consisting ofchloro, bromo, iodo and nitro; the acid addition salts thereof and thepharmaceutically acceptable metal salts thereof.

11. A compound selected from the group consisting of compounds of theformula:

(IZIVHX N(CH3)2 ON I OH, X CONE:

wherein X is selected from the group consisting of chloro, bromo andiodo; and X is selected from the group consisting of chloro, bromo,iodo, nitro and amino; the acid addition salts thereof and thepharmaceutically acceptable metal salts thereof. I

12. 9-bromo-6-deoxy-6-demethyl-6-bromomethylenetetracycline.

25 13. 9-chloro-6-deoxy-6-demethyl-6-ch1oromethylenetetracycline. v

14. A compound selected from the group consisting of compounds of theformula:

o ONE; 5 Y II II H 0 0 wherein X is selected from the group consistingof chloro, bromo, iodo and nitro and Y is selected from the groupconsisting of chloro and fluoro; the acid addition salts 17. A compoundselected from the group consisting of compounds of the formula:

OHX

Xi CONH! 6H 2% Y t) 6 wherein X is selected from the group consisting ofchloro, bromo and iodo; X is selected from the group consisting ofchloro, bromo, iodo and nitro; and Y is selected from the groupconsisting of chloro and fluoro; the acid addition salts thereof and thepharmaceutically acceptable metal salts thereof.

18. 11a-chloro-9-bromo-6-de0xy-6-demethyl-6-bromomethylenetetracycline.

19. 9,1 1a-dichloro-6-deoxy-6-demethyl-6-chloromethylenetetracycline.

No references cited.

HENRY R. JILES, Acting Primary Examiner.

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF COMPOUNDS OF THEFORMULAE:
 7. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF COMPOUNDSOF THE FORMULAE: